[{"id":443404,"pmid":26196957,"pmcid":null,"title":"Suppression of Poly(rC)-Binding Protein 4 (PCBP4) reduced cisplatin resistance in human maxillary cancer cells.","year":2015,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"Cisplatin plays an important role in the therapy for human head and neck cancers. However, cancer cells develop cisplatin resistance, leading to difficulty in treatment and poor prognosis. To analyze cisplatin-resistant mechanisms, a cisplatin-resistant cell line, IMC-3CR, was established from the IMC-3 human maxillary cancer cell line. Flow cytometry revealed that, compared with IMC-3 cells, cisplatin more dominantly induced cell cycle G2/M arrest rather than apoptosis in IMC-3CR cells. That fact suggests that IMC-3CR cells avoid cisplatin-induced apoptosis through induction of G2/M arrest, which allows cancer cells to repair damaged DNA and survive. In the present study, we specifically examined Poly(rC)-Binding Protein 4 (PCBP4), which reportedly induces G2/M arrest. Results showed that suppression of PCBP4 by RNAi reduced cisplatin-induced G2/M arrest and enhanced apoptosis in IMC-3CR cells, resulting in the reduction of cisplatin resistance. In contrast, overexpression of PCBP4 in IMC-3 cells induced G2/M arrest after cisplatin treatment and enhanced cisplatin resistance. We revealed that PCBP4 combined with Cdc25A and suppressed the expression of Cdc25A, resulting in G2/M arrest. PCBP4 plays important roles in the induction of cisplatin resistance in human maxillary cancers. PCBP4 is a novel molecular target for the therapy of head and neck cancers, especially cisplatin-resistant cancers.","journal":null,"figures":[],"_authors":null},{"id":1445,"pmid":22658674,"pmcid":null,"title":"Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.","year":2012,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed \"interactome capture,\" to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.","journal":null,"figures":[],"_authors":null},{"id":443403,"pmid":20817677,"pmcid":null,"title":"The cyclin-dependent kinase inhibitor p21 is regulated by RNA-binding protein PCBP4 via mRNA stability.","year":2011,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"RNA-binding proteins (RBPs) play a major role in many post-transcriptional processes, including mRNA stability, alternative splicing and translation. PCBP4, also called MCG10, is an RBP belonging to the poly(C)-binding protein family and a target of p53 tumor suppressor. Ectopic expression of PCBP4 induces cell-cycle arrest in G? and apoptosis. To identify RNA targets regulated by PCBP4 and further decipher its function, we generated multiple cell lines in which PCBP4 is either inducibly over-expressed or knocked down. We found that PCBP4 expression decreases cyclin-dependent kinase inhibitor p21 induction in response to DNA damage. We also provided evidence that PCBP4 regulates p21 expression independently of p53. In addition, we showed that a deficiency in PCBP4 enhances p21 induction upon DNA damage. To validate PCBP4 regulation of p21, we made PCBP4-deficient mice and showed that p21 expression is markedly increased in PCBP4-deficient primary mouse embryo fibroblasts compared to that in wild-type counterparts. Finally, we uncovered that PCBP4 binds to the 3'-UTR of p21 transcript in vitro and in vivo to regulate p21 mRNA stability. Taken together, we revealed that PCBP4 regulates both basal and stress-induced p21 expression through binding p21 3'-UTR and modulating p21 mRNA stability.","journal":null,"figures":[],"_authors":null},{"id":265,"pmid":20628086,"pmcid":null,"title":"Variation at the NFATC2 locus increases the risk of thiazolidinedione-induced edema in the Diabetes REduction Assessment with ramipril and rosiglitazone Medication (DREAM) study.","year":2010,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"Thiazolidinediones are used to treat type 2 diabetes. Their use has been associated with peripheral edema and congestive heart failure-outcomes that may have a genetic etiology.","journal":null,"figures":[],"_authors":null},{"id":254,"pmid":19913121,"pmcid":null,"title":"Gene-centric association signals for lipids and apolipoproteins identified via the HumanCVD BeadChip.","year":2009,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"Blood lipids are important cardiovascular disease (CVD) risk factors with both genetic and environmental determinants. The Whitehall II study (n=5592) was genotyped with the gene-centric HumanCVD BeadChip (Illumina). We identified 195 SNPs in 16 genes/regions associated with 3 major lipid fractions and 2 apolipoprotein components at p<10(-5), with the associations being broadly concordant with prior genome-wide analysis. SNPs associated with LDL cholesterol and apolipoprotein B were located in LDLR, PCSK9, APOB, CELSR2, HMGCR, CETP, the TOMM40-APOE-C1-C2-C4 cluster, and the APOA5-A4-C3-A1 cluster; SNPs associated with HDL cholesterol and apolipoprotein AI were in CETP, LPL, LIPC, APOA5-A4-C3-A1, and ABCA1; and SNPs associated with triglycerides in GCKR, BAZ1B, MLXIPL, LPL, and APOA5-A4-C3-A1. For 48 SNPs in previously unreported loci that were significant at p<10(-4) in Whitehall II, in silico analysis including the British Women's Heart and Health Study, BRIGHT, ASCOT, and NORDIL studies (total n>12,500) revealed previously unreported associations of SH2B3 (p<2.2x10(-6)), BMPR2 (p<2.3x10(-7)), BCL3/PVRL2 (flanking APOE; p<4.4x10(-8)), and SMARCA4 (flanking LDLR; p<2.5x10(-7)) with LDL cholesterol. Common alleles in these genes explained 6.1%-14.7% of the variance in the five lipid-related traits, and individuals at opposite tails of the additive allele score exhibited substantial differences in trait levels (e.g., >1 mmol/L in LDL cholesterol [approximately 1 SD of the trait distribution]). These data suggest that multiple common alleles of small effect can make important contributions to individual differences in blood lipids potentially relevant to the assessment of CVD risk. These genes provide further insights into lipid metabolism and the likely effects of modifying the encoded targets therapeutically.","journal":null,"figures":[],"_authors":null},{"id":443402,"pmid":17973258,"pmcid":null,"title":"Expression of alpha CP-4 inhibits cell cycle progression and suppresses tumorigenicity of lung cancer cells.","year":2008,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"The protein alpha CP-4 (also known as hnRNP E4) is an RNA binding protein encoded by a gene at 3p21, one of the most common altered regions in lung cancer. It has been proposed that alpha CP-4 may function as a lung tumor suppressor. Lack of alpha CP-4 expression is frequent in highly proliferative lung tumors and correlates with alpha CP-4 allele losses. The aim of this study was to evaluate the effect of alpha CP-4 on the tumorigenic capacity of lung cancer cells. alpha CP-4 expression was induced by transient transfection or stable infection with recombinant retroviruses. Induction of alpha CP-4 expression caused cell cycle arrest in G(2)/M in 3 out of the 7 lung cancer cell lines studied, while no effect on apoptosis was observed. Anchorage-independent growth and invasion capacity of H1299 cells were significantly reduced by alpha CP-4 induction. Tumorigenicity of H1299 cells in nude mice was greatly inhibited by the expression of alpha CP-4. Moreover, induction of alpha CP-4 expression in already established tumors resulted in a sudden growth arrest. Immunocytochemistry analysis of the xenograft tumors revealed an in vivo effect of alpha CP-4 on cell proliferation and no effect on apoptosis. Finally, alpha CP-4 showed a subcellular localization different from alpha CP-4a, a splice variant that does not affect cell proliferation. In conclusion, expression of alpha CP-4 can inhibit proliferation and tumorigenesis of lung cancer cells, both in vivo and in vitro, by delaying the progression of the cell cycle.","journal":null,"figures":[],"_authors":null},{"id":3038,"pmid":16713569,"pmcid":null,"title":"A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration.","year":2006,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"Many human inherited neurodegenerative disorders are characterized by loss of balance due to cerebellar Purkinje cell (PC) degeneration. Although the disease-causing mutations have been identified for a number of these disorders, the normal functions of the proteins involved remain, in many cases, unknown. To gain insight into the function of proteins involved in PC degeneration, we developed an interaction network for 54 proteins involved in 23 inherited ataxias and expanded the network by incorporating literature-curated and evolutionarily conserved interactions. We identified 770 mostly novel protein-protein interactions using a stringent yeast two-hybrid screen; of 75 pairs tested, 83% of the interactions were verified in mammalian cells. Many ataxia-causing proteins share interacting partners, a subset of which have been found to modify neurodegeneration in animal models. This interactome thus provides a tool for understanding pathogenic mechanisms common for this class of neurodegenerative disorders and for identifying candidate genes for inherited ataxias.","journal":null,"figures":[],"_authors":null},{"id":5,"pmid":16344560,"pmcid":null,"title":"Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.","year":2006,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.","journal":null,"figures":[],"_authors":null},{"id":4,"pmid":15489334,"pmcid":null,"title":"The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).","year":2004,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.","journal":null,"figures":[],"_authors":null},{"id":443401,"pmid":15205328,"pmcid":null,"title":"Alpha CP-4, encoded by a putative tumor suppressor gene at 3p21, but not its alternative splice variant alpha CP-4a, is underexpressed in lung cancer.","year":2004,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"alpha CP-4 is an RNA-binding protein coded by PCBP4, a gene mapped to 3p21, a common deleted region in lung cancer. In this study we characterized the expression of alpha CP-4 and alpha CP-4a, an alternatively spliced variant of alpha CP-4, in lung cancer cell lines and non-small cell lung cancer (NSCLC) samples from early stage lung cancer patients. In NSCLC biopsies, an immunocytochemical analysis showed cytoplasmic expression of alpha CP-4 and alpha CP-4a in normal lung bronchiolar epithelium. In contrast, alpha CP-4 immunoreactivity was not found in 47% adenocarcinomas and 83% squamous cell carcinomas, whereas all of the tumors expressed alpha CP-4a. Besides, lack of alpha CP-4 expression was associated with high proliferation of the tumor (determined by Ki67 expression). By fluorescence in situ hybridization, >30% of NSCLC cell lines and tumors showed allelic losses at PCBP4, correlating with the absence of the protein. On the other hand, no mutations in the coding region of the gene were found in any of the 24 cell lines analyzed. By Northern blotting and real-time reverse transcription-PCR, we detected the expression of alpha CP-4 and alpha CP-4a messages in NSCLC and small cell lung cancer cell lines. Our data demonstrate an abnormal expression of alpha CP-4 in lung cancer, possibly associated with an altered processing of the alpha CP-4 mRNA leading to a predominant expression of alpha CP-4a. This may be considered as an example of alternative splicing involved in tumor suppressor gene inactivation. Finally, induction of alpha CP-4 expression reduced cell growth, in agreement with its proposed role as a tumor suppressor, and suggesting an association of this RNA-binding protein with lung carcinogenesis.","journal":null,"figures":[],"_authors":null},{"id":3,"pmid":14702039,"pmcid":null,"title":"Complete sequencing and characterization of 21,243 full-length human cDNAs.","year":2004,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"As a base for human transcriptome and functional genomics, we created the \"full-length long Japan\" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.","journal":null,"figures":[],"_authors":null},{"id":430264,"pmid":14612387,"pmcid":null,"title":"A novel set of nuclear localization signals determine distributions of the alphaCP RNA-binding proteins.","year":2003,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"AlphaCPs comprise a subfamily of KH-domain-containing RNA-binding proteins with specificity for C-rich pyrimidine tracts. These proteins play pivotal roles in a broad spectrum of posttranscriptional events. The five major alphaCP isoforms are encoded by four dispersed loci. Each isoform contains three repeats of the RNA-binding KH domain (KH1, KH2, and KH3) but lacks other identifiable motifs. To explore the complexity of their respective functions, we examined the subcellular localization of each alphaCP isoform. Immunofluorescence studies revealed three distinct distributions: alphaCP1 and alphaCP2 are predominantly nuclear with specific enrichment of alphaCP1 in nuclear speckles, alphaCP3 and alphaCP4 are restricted to the cytoplasm, and alphaCP2-KL, an alphaCP2 splice variant, is present at significant levels in both the nucleus and the cytoplasm. We mapped nuclear localization signals (NLSs) for alphaCP isoforms. alphaCP2 contains two functionally independent NLS. Both NLSs appear to be novel and were mapped to a 9-amino-acid segment between KH2 and KH3 (NLS I) and to a 12-amino-acid segment within KH3 (NLS II). NLS I is conserved in alphaCP1, whereas NLS II is inactivated by two amino acid substitutions. Neither NLS is present in alphaCP3 or alphaCP4. Consistent with mapping studies, deletion of NLS I from alphaCP1 blocks its nuclear accumulation, whereas NLS I and NLS II must both be inactivated to block nuclear accumulation of alphaCP2. These data demonstrate an unexpected complexity in the compartmentalization of alphaCP isoforms and identify two novel NLS that play roles in their respective distributions. This complexity of alphaCP distribution is likely to contribute to the diverse functions mediated by this group of abundant RNA-binding proteins.","journal":null,"figures":[],"_authors":null},{"id":2,"pmid":12477932,"pmcid":null,"title":"Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.","year":2002,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:mgc.nci.nih.gov).","journal":null,"figures":[],"_authors":null},{"id":443392,"pmid":12003487,"pmcid":null,"title":"The poly(C)-binding proteins: a multiplicity of functions and a search for mechanisms.","year":2002,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"The poly(C) binding proteins (PCBPs) are encoded at five dispersed loci in the mouse and human genomes. These proteins, which can be divided into two groups, hnRNPs K/J and the alphaCPs (alphaCP1-4), are linked by a common evolutionary history, a shared triple KH domain configuration, and by their poly(C) binding specificity. Given these conserved characteristics it is remarkable to find a substantial diversity in PCBP functions. The roles of these proteins in mRNA stabilization, translational activation, and translational silencing suggest a complex and diverse set of post-transcriptional control pathways. Their additional putative functions in transcriptional control and as structural components of important DNA-protein complexes further support their remarkable structural and functional versatility. Clearly the identification of additional binding targets and delineation of corresponding control mechanisms and effector pathways will establish highly informative models for further exploration.","journal":null,"figures":[],"_authors":null},{"id":22653,"pmid":11984006,"pmcid":null,"title":"The Chediak-Higashi protein interacts with SNARE complex and signal transduction proteins.","year":2002,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this study was to identify proteins that interact with LYST as a first step in understanding how LYST modulates lysosomal exocytosis.","journal":null,"figures":[],"_authors":null},{"id":443400,"pmid":11313928,"pmcid":null,"title":"Regulation of the G2/M transition by p53.","year":2001,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"p53 protects mammals from neoplasia by inducing apoptosis, DNA repair and cell cycle arrest in response to a variety of stresses. p53-dependent arrest of cells in the G1 phase of the cell cycle is an important component of the cellular response to stress. Here we review recent evidence that implicates p53 in controlling entry into mitosis when cells enter G2 with damaged DNA or when they are arrested in S phase due to depletion of the substrates required for DNA synthesis. Part of the mechanism by which p53 blocks cells at the G2 checkpoint involves inhibition of Cdc2, the cyclin-dependent kinase required to enter mitosis. Cdc2 is inhibited simultaneously by three transcriptional targets of p53, Gadd45, p21, and 14-3-3 sigma. Binding of Cdc2 to Cyclin B1 is required for its activity, and repression of the cyclin B1 gene by p53 also contributes to blocking entry into mitosis. p53 also represses the cdc2 gene, to help ensure that cells do not escape the initial block. Genotoxic stress also activates p53-independent pathways that inhibit Cdc2 activity, activation of the protein kinases Chk1 and Chk2 by the protein kinases Atm and Atr. Chk1 and Chk2 inhibit Cdc2 by inactivating Cdc25, the phosphatase that normally activates Cdc2. Chk1, Chk2, Atm and Atr also contribute to the activation of p53 in response to genotoxic stress and therefore play multiple roles. p53 induces transcription of the reprimo, B99, and mcg10 genes, all of which contribute to the arrest of cells in G2, but the mechanisms of cell cycle arrest by these genes is not known. Repression of the topoisomerase II gene by p53 helps to block entry into mitosis and strengthens the G2 arrest. In summary, multiple overlapping p53-dependent and p53-independent pathways regulate the G2/M transition in response to genotoxic stress.","journal":null,"figures":[],"_authors":null},{"id":430261,"pmid":10936052,"pmcid":null,"title":"Identification of two novel mammalian genes establishes a subfamily of KH-domain RNA-binding proteins.","year":2000,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"We have identified two novel human genes encoding proteins with a high level of sequence identity to two previously characterized RNA-binding proteins, alphaCP-1 and alphaCP-2. Both of these novel genes, alphaCP-3 and alphaCP-4, are predicted to encode proteins with triplicated KH domains. The number and organization of the KH domains, their sequences, and the sequences of the contiguous regions are conserved among all four alphaCP proteins. The common evolutionary origin of these proteins is substantiated by conservation of exon-intron organization in the corresponding genes. The map positions of alphaCP-1 and alphaCP-2 (previously reported) and those of alphaCP-3 and alphaCP-4 (present report) reveal that the four alphaCP loci are dispersed in the human genome; alphaCP-3 and alphaCP-4 mapped to 21q22.3 and 3p21, and the respective mouse orthologues mapped to syntenic regions of the mouse genome, 10B5 and 9F1-F2, respectively. Two additional loci in the human genome were identified as alphaCP-2 processed pseudogenes (PCBP2P1, 21q22.3, and PCBP2P2, 8q21-q22). Although the overall levels of alphaCP-3 and alphaCP-4 mRNAs are substantially lower than those of alphaCP-1 and alphaCP-2, transcripts of alphaCP-3 and alphaCP-4 were found in all mouse tissues tested. These data establish a new subfamily of genes predicted to encode closely related KH-containing RNA-binding proteins with potential functions in posttranscriptional controls.","journal":null,"figures":[],"_authors":null},{"id":443399,"pmid":10891498,"pmcid":null,"title":"MCG10, a novel p53 target gene that encodes a KH domain RNA-binding protein, is capable of inducing apoptosis and cell cycle arrest in G(2)-M.","year":2000,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"p53, a tumor suppressor, inhibits cell proliferation by inducing cellular genes involved in the regulation of the cell cycle. MCG10, a novel cellular p53 target gene, was identified in a cDNA subtraction assay with mRNA isolated from a p53-producing cell line. MCG10 can be induced by wild-type but not mutant p53 and by DNA damage via two potential p53-responsive elements in the promoter of the MCG10 gene. The MCG10 gene contains 10 exons and is located at chromosome 3p21, a region highly susceptible to aberrant chromosomal rearrangements and deletions in human neoplasia. The MCG10 gene locus encodes at least two alternatively spliced transcripts, MCG10 and MCG10as. The MCG10 and MCG10as proteins contain two domains homologous to the heterogeneous nuclear ribonucleoprotein K homology (KH) domain. By generating cell lines that inducibly express either wild-type or mutated forms of MCG10 and MCG10as, we found that MCG10 and MCG10as can suppress cell proliferation by inducing apoptosis and cell cycle arrest in G(2)-M. In addition, we found that MCG10 and MCG10as, through their KH domains, can bind poly(C) and that their RNA-binding activity is necessary for inducing apoptosis and cell cycle arrest. Furthermore, we found that the level of the poly(C) binding MCG10 protein is increased in cells treated with the DNA-damaging agent camptothecin in a p53-dependent manner. These results suggest that the MCG10 RNA-binding protein is a potential mediator of p53 tumor suppression.","journal":null,"figures":[],"_authors":null},{"id":548,"pmid":8889548,"pmcid":null,"title":"Normalization and subtraction: two approaches to facilitate gene discovery.","year":1996,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"Large-scale sequencing of cDNAs randomly picked from libraries has proven to be a very powerful approach to discover (putatively) expressed sequences that, in turn, once mapped, may greatly expedite the process involved in the identification and cloning of human disease genes. However, the integrity of the data and the pace at which novel sequences can be identified depends to a great extent on the cDNA libraries that are used. Because altogether, in a typical cell, the mRNAs of the prevalent and intermediate frequency classes comprise as much as 50-65% of the total mRNA mass, but represent no more than 1000-2000 different mRNAs, redundant identification of mRNAs of these two frequency classes is destined to become overwhelming relatively early in any such random gene discovery programs, thus seriously compromising their cost-effectiveness. With the goal of facilitating such efforts, previously we developed a method to construct directionally cloned normalized cDNA libraries and applied it to generate infant brain (INIB) and fetal liver/spleen (INFLS) libraries, from which a total of 45,192 and 86,088 expressed sequence tags, respectively, have been derived. While improving the representation of the longest cDNAs in our libraries, we developed three additional methods to normalize cDNA libraries and generated over 35 libraries, most of which have been contributed to our integrated Molecular Analysis of Genomes and Their Expression (IMAGE) Consortium and thus distributed widely and used for sequencing and mapping. In an attempt to facilitate the process of gene discovery further, we have also developed a subtractive hybridization approach designed specifically to eliminate (or reduce significantly the representation of) large pools of arrayed and (mostly) sequenced clones from normalized libraries yet to be (or just partly) surveyed. Here we present a detailed description and a comparative analysis of four methods that we developed and used to generate normalize cDNA libraries from human (15), mouse (3), rat (2), as well as the parasite Schistosoma mansoni (1). In addition, we describe the construction and preliminary characterization of a subtracted liver/spleen library (INFLS-SI) that resulted from the elimination (or reduction of representation) of -5000 INFLS-IMAGE clones from the INFLS library.","journal":null,"figures":[],"_authors":null},{"id":1540,"pmid":8125298,"pmcid":null,"title":"Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.","year":1994,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).","journal":null,"figures":[],"_authors":null}]